Abstract: Sensory hair cells are required for balance function. Vestibular hair cell degeneration causes balance dysfunction/hypofunction manifested as dizziness and vertigo. While the mammalian cochlea lacks the ability to regenerate lost hair cells, a limited degree of spontaneous regeneration occurs in the utricle, a vestibular organ detecting linear acceleration. Recent studies using fate-mapping techniques have pinpointed supporting cells as precursors of regenerated hair cells. However, it is not clear whether regenerated hair cells are fully functional and if organ function recovers. In preliminary experiments we have characterized hair cell degeneration and regeneration in the mature mouse utricle and also a loss followed by recovery of vestibular evoked potentials (VsEP) in vivo. The first aim of this proposal is to determine if increasing hair cell regeneration improves the recovery of vestibular function. Specifically, regenerated hair cells labeled via fate-mapping are probed via histology and electrophysiology to assess bundle morphology, mechanosensitvity, basolateral currents, and synaptic properties including vesicle release. In parallel, VsEP responses are measured and compared to histologic and electrophysiological measures. Next, by overexpressing Atoh1 via a transgenic approach, we will study the histology and electrophysiology of Atoh1-overexpressing hair cells and also the overall VsEP responses. In the second aim, we will determine if Atoh1 deletion prevents hair cell regeneration and the recovery of VsEP responses. In parallel, fate-mapped, surviving hair cells will be examined for possible repair via histology and electrophysiology. To gain an unbiased insight into the genetic signature of hair cell progenitors and surviving hair cells, the third aim is designed to examine the damaged mature mouse utricle using single cell RNA sequencing technologies. Here the first goal is to discover the genetic landscape of hair cell progenitors and surviving hair cells in the damaged utricle. Secondly, we will examine the gene expression of the undamaged and damaged utricle after Atoh1 overexpression. Lastly, we will use bioinformatic approaches to delineate the trajectory of the spontaneous and Atoh1-enhaced supporting cell-hair cell transition and validate this histologically. In summary, we will apply state-of-the art technologies (vestibular physiology, hair cell physiology, single cell RNA-seq, bioinformatic strategies) to study vestibular hair cell regeneration in transgenic mouse models. We have assembled a team of experts who have worked together to collect promising preliminary data. At the end of this 5-year proposal, we will have 1) determined the relationship between hair cell regeneration and functional recovery and 2) revealed and temporally ordered novel genes during mammalian hair cell regeneration.